Improved gene therapy constructs for the treatment of propionic acidemia caused by mutations in propionyl-coa carboxylase alpha

Abstract

Polynucleotide expression cassettes comprising synthetic polynucleotides encoding human propionyl-CoA carboxylase alpha (synPCCA) are described herein. Related recombinant expression vectors, recombinant adeno-associated viruses (rAAVs), and compositions are also described. Also described are methods of treating a disease or condition mediated by propionyl-CoA carboxylase, comprising administering to a subject in need thereof a therapeutic amount of any of the polynucleotide expression cassettes, recombinant expression vectors, rAAVs, or compositions.

1 . A polynucleotide expression cassette comprising:
 (a) synthetic propionyl-CoA carboxylase subunit a (PCCA) polynucleotide (synPCCA) selected from the group consisting of:
 (i) a polynucleotide comprising the nucleic acid sequence of any one of SEQ ID NOs: 2-7; and 
 (ii) a polynucleotide comprising a nucleic acid sequence with at least 80% identity to the nucleic acid sequence of any one of SEQ ID NOs: 2-7 which encodes the polypeptide of SEQ ID NO:8 and has equivalent or greater expression in a host relative to expression of any one of SEQ ID NOs: 2-7 or SEQ ID NO:1, wherein the polynucleotide does not have the nucleic acid sequence of SEQ ID NO: 1; and 
   (b) any one or more of the following features:
 (i) a 3′ inverted terminal repeat (3′ ITR) with a length greater than 135 base pairs (bp) and less than 145 bp; 
 (ii) fewer CpG dinucleotides as compared to a nucleic acid sequence of any one or more of SEQ ID NOs: 9-12 and 35-40; and 
 (iii) fewer restriction enzyme recognition sites as compared to a nucleic acid sequence of any one or more of SEQ ID NOs: 9-12 and 35-40. 
   
     
     
         2 . The polynucleotide expression cassette of  claim 1 , further comprising any one or more of the following features:
 (i) a 5′ ITR nucleotide sequence at least 90% identical to nucleotides 1 to 130 of SEQ ID NO: 41;   (ii) a core elongation factor 1 alpha (EF1S) promoter nucleotide sequence at least 90% identical to nucleotides 131 to 361 of SEQ ID NO: 41;   (iii) an intron nucleotide sequence at least 90% identical to nucleotides 362 to 526 of SEQ ID NO: 41;   (iv) a hepatitis B post translation response element (HPRE) nucleotide sequence at least 90% identical to nucleotides 2714 to 3439 of SEQ ID NO: 41;   (v) a polyA nucleotide sequence at least 90% identical to nucleotides 3440 to 3664 of SEQ ID NO: 41; and   (vi) a 3′ ITR nucleotide sequence at least 90% identical to nucleotides 3665 to 3805 of SEQ ID NO: 41.   
     
     
         3 . The polynucleotide expression cassette of  claim 1 , further comprising all of the following features:
 (i) a 5′ ITR nucleotide sequence at least 90% identical to nucleotides 1 to 130 of SEQ ID NO: 41;   (ii) a EF1S promoter nucleotide sequence at least 90% identical to nucleotides 131 to 361 of SEQ ID NO: 41;   (iii) an intron nucleotide sequence at least 90% identical to nucleotides 362 to 526 of SEQ ID NO: 41;   (iv) a HPRE nucleotide sequence at least 90% identical to nucleotides 2714 to 3439 of SEQ ID NO: 41;   (v) a polyA nucleotide sequence at least 90% identical to nucleotides 3440 to 3664 of SEQ ID NO: 41; and   (vi) a 3′ ITR nucleotide sequence at least 90% identical to nucleotides 3665 to 3805 of SEQ ID NO: 41.   
     
     
         4 . The polynucleotide expression cassette of  claim 1 , wherein the synPCCA polynucleotide comprises a synPCCA1 nucleotide sequence at least 90% identical to SEQ ID NO: 2. 
     
     
         5 . The polynucleotide expression cassette of  claim 1 , comprising any one or more of the following features:
 (i) a 5′ ITR nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 1 to 130 of SEQ ID NO: 41;   (ii) a EF1S promoter nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 131 to 361 of SEQ ID NO: 41;   (iii) a intron nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 362 to 526 of SEQ ID NO: 41;   (iv) a HPRE nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 2714 to 3439 of SEQ ID NO: 41;   (v) a polyA nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 3440 to 3664 of SEQ ID NO: 41; and   (vi) a 3′ ITR nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 3665 to 3805 of SEQ ID NO: 41.   
     
     
         6 . The polynucleotide expression cassette of  claim 1 , comprising all of the following features:
 (i) a 5′ ITR nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 1 to 130 of SEQ ID NO: 41;   (ii) a EF1S promoter nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 131 to 361 of SEQ ID NO: 41;   (iii) a intron nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 362 to 526 of SEQ ID NO: 41;   (iv) a HPRE nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 2714 to 3439 of SEQ ID NO: 41;   (v) a polyA nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 3440 to 3664 of SEQ ID NO: 41; and   (vi) a 3′ ITR nucleotide sequence consisting of a nucleotide sequence at least 90% identical to nucleotides 3665 to 3805 of SEQ ID NO: 41.   
     
     
         7 . The polynucleotide expression cassette of  claim 1 , wherein the synPCCA polynucleotide consists of a synPCCA1 nucleotide sequence at least 90% identical to SEQ ID NO: 2. 
     
     
         8 . The polynucleotide expression cassette of  claim 1 , comprising a nucleotide sequence at least 90% identical to SEQ ID NO: 41 or SEQ ID NO: 42. 
     
     
         9 . The polynucleotide expression cassette of  claim 1 , consisting of a nucleotide sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 41 or SEQ ID NO: 42. 
     
     
         10 . A recombinant expression vector comprising the polynucleotide expression cassette of  claim 1 . 
     
     
         11 . The recombinant expression vector of  claim 10 , wherein the vector is a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises an AAV capsid and a vector genome packaged therein. 
     
     
         12 . The rAAV according to  claim 11 , wherein the vector is comprised of the structure in  FIG.  15 B . 
     
     
         13 . The rAAV according to  claim 11 , wherein the AAV capsid is from an AAV of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh 10, hu37 or Anc, or mutants thereof. 
     
     
         14 . The rAAV according to  claim 13 , wherein the AAV capsid is from an AAV of serotype 9. 
     
     
         15 . A composition comprising (a)(i) the polynucleotide expression cassette of  claim 1  or (ii) a recombinant expression vector comprising the polynucleotide expression cassette of (i), and (b) a pharmaceutically acceptable carrier. 
     
     
         16 . A method for treating a disease or condition mediated by propionyl-CoA carboxylase in a subject, the method comprising administering (i) the polynucleotide expression cassette of  claim 1 , (ii) a recombinant expression vector comprising the polynucleotide expression cassette of (i, or (iii) a composition comprising (i) or (ii) and a pharmaceutically acceptable carrier to a subject, wherein the subject has a disease or condition mediated by propionyl-CoA carboxylase. 
     
     
         17 . The method of  claim 16 , wherein the disease or condition is propionic acidemia (PA). 
     
     
         18 . The method of  claim 16 , wherein the vector is an rAAV and is formulated for administration at a dose of 1×10 11  to 1×10 14  genome copies (GC)/kg. 
     
     
         19 . The method of  claim 16 , wherein the vector is an rAAV and is formulated for administration as a single dose of rAAV. 
     
     
         20 . The method of  claim 16 , wherein the vector is an rAAV and is formulated for administration as multiple doses of rAAV. 
     
     
         21 . An in vitro method comprising administering to a cell of a subject, or a population of cells of the subject, the polynucleotide expression cassette of  claim 1 , wherein the polynucleotide expression cassette is inserted into the cell of the subject, or the population of cells of the subject, via genome editing on the cell of the subject using a nuclease selected from the group of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the clustered regularly interspaced short palindromic repeats (CRISPR/cas system) and meganuclease re-engineered homing endonucleases on the cell from the subject, or the population of cells of the subject. 
     
     
         22 . The method of  claim 16 , wherein the polynucleotide expression cassette, recombinant expression vector, rAAV, or composition is formulated for subcutaneous, intramuscular, intradermal, intraperitoneal, or intravenous administration. 
     
     
         23 . A nucleic acid consisting of (a) a nucleotide sequence that is at least 12 but no more than 35 contiguous nucleotides of any one of SEQ ID NO: 2-7; (b) a nucleotide sequence that is at least 90% identical to at least 12 but no more than 35 contiguous nucleotides of any one of SEQ ID NO: 2-7; or (c) a nucleotide sequence that is complementary to (a) or (b). 
     
     
         24 . The nucleic acid of  claim 23 , further comprising a detectable label. 
     
     
         25 . A collection of nucleic acids comprising two or more of the nucleic acids of  claim 23 . 
     
     
         26 . The collection according to  claim 25 , wherein the two or more nucleic acids comprise two or more different nucleic acids. 
     
     
         27 . An in vitro method for detecting the expression of synPCCA nucleic acid by cells, the method comprising:
 (a) contacting a sample comprising nucleic acid from the cells with the nucleic acid of  claim 23  or a collection comprising two or more thereof, thereby forming a complex; and   (b) detecting the complex, wherein detection of the complex is indicative of the expression of synPCCA nucleic acid by the cells.   
     
     
         28 . The method of  claim 27 , wherein the method is carried out prior to administering (i) the polynucleotide expression cassette of  claim 1 , (ii) a recombinant expression vector comprising the polynucleotide expression cassette of (i, or (iii) a composition comprising (i) or (ii) and a pharmaceutically acceptable carrier to a subject. 
     
     
         29 . The method of  claim 27 , wherein the cells are from a subject that has been treated with (i) the polynucleotide expression cassette of the polynucleotide expression cassette of  claim 1 , (ii) a recombinant expression vector comprising the polynucleotide expression cassette of (i), or (iii) a composition comprising (i) or (ii) and a pharmaceutically acceptable carrier, and wherein detection of the complex is indicative of the expression of synPCCA nucleic acid by the cells of the subject. 
     
     
         30 . The method of  claim 27 , wherein the cells are heart cells or liver cells. 
     
     
         31 . The method of  claim 27 , wherein the cells are hepatocytes or cardiomyocytes. 
     
     
         32 . The method of  claim 27 , wherein the cells are cells of a cell line. 
     
     
         33 . The method of  claim 27 , wherein the cells are cells of a heart cell line or a liver cell line. 
     
     
         34 . The method of  claim 27 , wherein the cells are cells of a hepatocyte cell line or a cardiomyocyte cell line. 
     
     
         35 . The method of  claim 34 , wherein the cells are cells of a hepatocyte cell line which does not express one or both of endogenous PCCA mRNA and PCCA protein. 
     
     
         36 . The method of  claim 27 , further comprising quantifying the synPCCA nucleic acid expressed by the cells. 
     
     
         37 . The method of  claim 27 , further comprising quantifying the cells that express the synPCCA nucleic acid.